Alpha-amylase variants with E188P and S242Y substitutions
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Solution Overview
Problem
Current alpha-amylase variants lack sufficient stability at low pH and low calcium concentrations, and do not efficiently produce a starch liquefact with optimal dextrose equivalent (DE) values for saccharification processes.
Innovation Solution
Alpha-amylase variants with specific substitutions at positions 188, 242, and 275, such as E188P combined with S242Y, F, H, W, P, I, T, L, K279Y, F, H, W, I, D, M, S, N, Q, V, A, and N275Y, F, H, W, which enhance thermo-stability and chelator stability, are developed.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If alpha-amylase variants are designed to improve stability at low pH and low calcium concentrations, then thermo-stability and chelator stability are enhanced, but the enzyme may lose catalytic activity or saccharification efficiency
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid residues at positions 188, 242, 275, and 279 to optimize the balance between stability and catalytic activity. Specific substitutions (e.g., E188P combined with S242Y/F/H/W/P/I/T/L, K279Y/F/H/W/I/D/M/S/N/Q/V/A, N275Y/F/H/W) were identified to enhance thermo-stability and chelator stability while maintaining DE values in the optimal range of 10-16 for efficient saccharification
Solution Approach 2:
The patent applies local quality by making targeted amino acid substitutions at specific positions (188, 242, 275, 279) rather than global modifications. Each position was independently optimized to provide local structural improvements that enhance stability without compromising overall catalytic function. The combinations of substitutions at these specific positions create localized structural changes that resolve the stability-activity trade-off
2Productivity
If alpha-amylase variants are engineered to produce liquefact with optimal DE values (10-16) for efficient saccharification, then productivity is improved, but the enzyme may exhibit reduced stability under process conditions
Solution Approach 1:
The patent simultaneously optimizes multiple parameters including amino acid substitutions at positions 188, 242, 275, and 279, pH conditions (4.5-5.0), temperature (80-90°C), and calcium concentration (0.12 mM) to achieve both high productivity with DE values of 10-16 and enhanced stability with T1/2 of at least 10 minutes under process conditions
Solution Approach 2:
The patent applies preliminary action by pre-optimizing the enzyme structure through specific amino acid substitutions before the liquefaction process. The variants are engineered in advance with substitutions at positions 188, 242, 275, and 279 to ensure they maintain stability and produce optimal DE values (10-16) during the subsequent saccharification process, eliminating the need for process adjustments
3Reliability
If multiple amino acid substitutions are introduced to enhance both thermo-stability and chelator stability, then reliability is improved, but the complexity of enzyme production and characterization increases
Solution Approach 1:
The patent applies local quality by focusing mutations on four specific positions (188, 242, 275, 279) rather than random or global modifications. This localized approach to protein engineering simplifies the production and characterization process compared to comprehensive mutagenesis, as it requires testing only specific substitution combinations at defined positions rather than screening numerous random variants
Solution Approach 2:
The patent systematically evaluates specific substitution combinations at positions 188, 242, 275, and 279 to identify optimal variants. By predefined substitution options (e.g., E188P with specific residues at 242, 279, 275), the patent reduces the complexity of enzyme production and characterization while achieving enhanced chelator stability and thermo-stability
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The variants exhibit improved stability at pH 4.5 and increased chelator stability, producing a liquefact with higher DE values, enhancing saccharification efficiency and stability compared to parent alpha-amylases.
Implementation Method 1
Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1) constitute a group of enzymes, which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides
Data Source
AI summary
The present invention relates to alpha-amylase variants comprising a substitution at a position corresponding to position 188 and at least one further substitution at a position corresponding to position 242 or 279 or 275 of SEQ ID NO: 1, in particular one or more combinations of substitutions selected from the group consisting of E188P+S242Y, E188P+S242F, E188P+S242H, E188P+S242W, E188P+S242P, E188P+S242I, E188P+S242T, E188P+S242L, E188P+K279W, E188P+K279Y, E188P+K279F, E188P+K279H, E188P+K279I, E188P+K279L, E188P+K279D, E188P+K279M, E188P+K279S, E188P+K279T E188P+K279N, E188P+K279Q, E188P+K279V, E188P+K279A, E188P+N275F, E188P+N275Y, E188P+N275W, and E188P+N275H, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to a parent alpha amylase selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 27. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.